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Cellular Signalling 17
Herpes virus proteins ICP0 and BICP0 can activate NF-nB by catalyzing
InBa ubiquitination
Lirong Diaoa,b, Bianhong Zhanga, Junkai Fana, Xiang Gaoa, Shaogang Suna, Kai Yanga,
Dan Xinb, Naihe Jina, Yunqi Gengb, Chen Wanga,*
aLaboratory of Molecular and Cellular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences, Box 49, 320 Yue Yang Road, Shanghai 200031, P.R. ChinabCollege of Life Science, Nan Kai University, Tianjin 300071, P.R. China
Received 4 July 2004; received in revised form 12 July 2004; accepted 12 July 2004
Available online 25 August 2004
Abstract
The immediate early proteins ICP0 and BICP0 from Herpes virus are promiscuous activators of both viral and cellular genes and play a
critical role in virus life cycle. Here we report that ICP0 and BICP0 could induce NF-nB translocation from cytoplasm into nucleus and
strongly activate NF-nB responsive genes specifically. This process was dependent on the RING domain of both proteins. In addition, ICP0
interacted specifically with InBa and its activating effect was attenuated by Ubch5A(C85A) and MG132, but not by InBa(S32A/S36A).Remarkably, InBa was poly-ubiquitinated by both ICP0 and BICP0, in vitro and in vivo. These data indicate that ICP0 and BICP0,
functioning as ubiquitin ligases, are bona fide activators of NF-nB signaling pathway. Our study identifies a new way ICP0 and BICP0
explore to regulate gene expression.
D 2004 Elsevier Inc. All rights reserved.
Keywords: Herpes virus; ICP0; BICP0; Ubiquitin; InBa
1. Introduction
Herpes simplex viruses (HSV) are nuclearly replicating,
icosahedral, enveloped DNA viruses. They are the culprits
of cold sores and other more serious diseases [1]. HSV-1 has
developed a successful strategy to establish a life-long latent
infection in the nervous system after initial entry into the
epithelial cells [2]. In response to stress, it can reactivate
into lytic cycle and express the immediate-early genes such
as ICP0, ICP4, ICP27, which then turn on the early and late
genes [3]. Remarkably, HSV-1 functional counterpart in
bovine has been characterized as bovine herpes virus 1
(BHV-1) that shares a number of biological properties with
HSV-1 in terms of life cycle and protein functions [4].
0898-6568/$ - see front matter D 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.cellsig.2004.07.003
* Corresponding author. Tel.: +86 21 54921185.
E-mail addresses: [email protected] (Y. Geng)8
[email protected] (C. Wang).
ICP0 is a 775-amino-acid multifunctional protein that is
expressed immediately after HSV-1 infection or reactivation
[5]. It is critical for the efficient initiation of lytic infection
and sufficient to induce HSV-1 reactivation from neuronal
latency [6]. The underlying mechanisms have been under
intensive study. Initially, ICP0 was found to promote both
viral and cellular protein production by stimulating mRNA
synthesis [7]. However, it failed to identify signature
promoter sequence recognized by ICP0; nor was it able to
demonstrate any affinity between ICP0 and DNA, which
suggested that the mode of ICP0 action was not a simple
case of direct transcriptional activation [5]. This conjecture
was partly substantiated by the observation that ICP0 co-
localized with the nuclear substructures called ND10 and
mediated the disruption of them [8–10]. Recently, ICP0 was
shown to interact specifically with a diverse collection of
proteins including USP7, cyclin D3, elongation factor EF-
1y, transcription factor BMAL1 and HSV-1 ICP4 [11–15].
Importantly, ICP0 contains a RING domain at its N-
(2005) 217–229
L. Diao et al. / Cellular Signalling 17 (2005) 217–229218
terminus and displays intrinsic ubiquitin ligase activity [16–
19]. In parallel, BICP0 is bovine homologue of ICP0 that
harbors a RING domain at its N-terminus and exhibits
ubiquitin ligase activity too (see data below).
Ubiquitin (Ub) is a 76-amino-acid globular protein that
plays an important role in regulating many aspects of
cellular processes, including NF-nB signaling pathway [20].
Conjugation of Ub onto other proteins requires the
sequential actions of three enzymes: Ub is first activated
in an ATP-dependent way by Ub activating enzyme (E1)
and then transferred to a Cys residue in the Ub-conjugating
enzyme (E2). With the help of Ub ligase (E3), Ub is
attached via its C-terminus to a q-amino group of the Lys
residues in the substrate proteins. Since Ub contains seven
lysines itself, poly-Ub chain can be formed by covalently
attaching one Ub to another Ub in the same way [21]. In
general, proteins with RING domain are members of the E3
family that are responsible for catalyzing ubiquitination of
target proteins [22]. Classically, Ub was regarded as a
bdeath-tagQ to target proteins for degradation by 26S
proteasome. However, new mechanisms of Ub actions have
been emerged and characterized [23,24].
Found in essentially all mammalian cell types, the
transcription factor NF-nB(p65/p50) regulates a wide rangeof genes important in inflammation, immunity, development
and apoptosis. It is normally sequestered in the cytoplasm
by virtue of its association with inhibitor InBa. A myriad of
stimuli can lead to the activation of InB kinase complex
(IKK) and subsequently phosphorylation of the serine
residues 32 and 36 in InBa. The phospho-InBa is then
ubiquitinated by a specific E3 called SCFh-TrCP and
degraded by the 26S proteasome. This releases NF-nBfrom its anchor, which then translocates into nucleus and
activates target gene expressions [25–29]. Interestingly,
ubiquitination was also used to regulate NF-nB signaling
pathway in other distinct ways [30].
Although it was known that manipulating NF-nB signal-
ing pathway could promote viral replication, host cell
survival or evasion of the immune response, different
viruses employed different mechanisms to do so [31].
Previously, it was demonstrated that ICP0 from HSV-1
could trans-activate the LTR promoter of HIV [32,33].
However, it remains unknown how ICP0 activates the LTR
promoter. Accidentally, we observed that stimulation by
BICP0 of the bovine immunodeficiency virus 1 (BIV-1)
enhancer LTR was severely impaired when a nB-consensusmotif within the LTR was deleted. In light of the facts that
ICP0 and BICP0 are promiscuous activator of gene
transcription, we hypothesized that ICP0 and BICP0 played
a key role in NF-nB activation. In this study, we
demonstrated that ICP0 and BICP0 could specifically
stimulate NF-nB responsive reporter gene expression in
different cell lines and this effect could be attenuated by
Ubch5A(C85A) and MG132. In addition, ICP0 and BICP0
could induce p65-NF-nB to translocate from cytoplasm into
nucleus and promote NF-nB DNA binding affinity.
Remarkably, ICP0 interacted with InBa and stimulated
InBa poly-ubiquitination in vitro and in vivo. However,
this process was independent of InBa phosphorylation. Our
data indicate that ICP0 and BICP0 are bona fide activators
of NF-nB signaling pathway and they function as ubiquitin
ligases to directly catalyze InBa ubiquitination.
2. Experimental procedure
2.1. Plasmids and reagents
cDNA constructs for ICP0, BICP0 and BICP0(13G/51A)
were gifts from Dr. Yange Zhang of University of Nebraska-
Lincoln. These cDNAs were subcloned into pDNA3.1-N-
Flag(a gift from Dr. Hai Wu in UT Southwestern Medical
Center). These constructs were expressed in mammalian
cells for functional analysis and immunoprecipitated with
anti-flag beads for in vitro assays. Truncating mutants of
ICP0 and BICP0 were made either by PCR or directly by
cutting out the corresponding segments and cloned into
pDNA3.1-N-Flag. The mutants covered one segment of the
full-length proteins: 1–234 aa for ICP0(exon1+2), 518–775
aa for ICP0(MC), 555–775 aa for ICP0(AC), 1–357 aa for
BICP0(N), 357–676 aa for BICP0(C). pcDNA3-HA-InBaand the truncating mutants were made by PCR of the
corresponding segments of human InBa and cloning them
into pcDNA3-HA plasmid (kindly provided by Professor
Gang Pei). GST-InBa was prepared by subcloning InBainto pGEX-4T-1 (Promega) and purified from E. coli by
Pharmacia kit. Radioactive InBa and the mutants were in
vitro translated using Promega TNT wheat germ system.
GST-ATF was kindly provided by Dr. Meng Li. All these
constructs were confirmed by automatic DNA sequencing.
The rest of the plasmids and proteins including the ubiquitin
system were prepared as described previously (Deng et.al,
2000) [24]. All antibodies were from Santa Cruz Biotech-
nology if not specified otherwise. From Aldrich-Sigma were
purchased anti-Flag bead, protein-A bead, anti-Ub mono-
clonal antibody and MG132, aprotinin, chymostatin, leu-
peptin. Radioactive isotopes were from Amersham
Biotechnology. The mouse polyclonal antibodies against
ICP0 and BICP0 were made by subcutaneously injecting 6-
week Kunming mice with the purified his-ICP0(C) and his-
BICP0 protein expressed from E. coli BL21. Both anti-
bodies recognized only their immunogens specifically.
2.2. Cell culture, transfection, and reporter gene assays
293T and SH-SY5Y cells were from ATCC, USA. HeLa
cells were kindly provided by professor Lin Li. These cells
were cultured in DMEM media supplemented with 10%
fetal bovine serum (FBS), penicillin (50 U/ml) and
streptomycin (50 Ag/ml). Cells (2.0�105 and 1.5�106)
were seeded in six-well plates for luciferase assay and 10-
cm plates for other experiments, respectively. Transfection
L. Diao et al. / Cellular Signalling 17 (2005) 217–229 219
with the indicated plasmids was carried out by either
calcium phosphate precipitation method [24] or lipofect-
amine 2000 (Invitrogen cat. no. 11668-027) according to the
manufacturer’s instructions. Cells were harvested 24–48 h
after transfection as indicated. For some experiments, cells
were treated with the indicated reagents 16 or 24 h after
transfection. For reporter gene assay, cells were harvested
48 h after transfection and gene activity was measured using
the Luciferase Assay System (Promega). Data were nor-
malized in reference to a GFP internal control. Each datum
was from a representative experiment reproducibly repeated
at least three times. The cell lysates for luciferase assay were
all checked with Western blot to ensure protein expression
of the various constructs. A plasmid expressing LacZ was
included in all transfection experiments as negative control.
2.3. Immunofluorescence and confocal microscopy
293T cells were seeded on chamber slides in 35-mm
plate and transiently transfected with 1 Ag of the
designated plasmids. After 16 h, cells were fixed at room
temperature with 4% (w/v) paraformaldehyde–PBS for 20
min and then permeabilized at room temperature by 0.5%
Triton X-100–PBS for 10 min. The cells were then
incubated at 37 8C with mouse anti-NF-nB p65(F-6)
monoclonal antibody for 1 h and then at 4 8C for 16 h.
After washing slides with 0.05% Triton X-100–PBS, the
cells were incubated with FITC conjugated anti-mouse
immunoglobulin G (IgG) for 1 h at 37 8C. The cells were
then co-stained with DAPI. The pictures were taken by
Bio-Rad confocal microscopy system Radiance 2100 with
Lasersharp 2000 software.
2.4. Preparation of soluble cell extracts
Infected or mock infected cells were rinsed in PBS twice,
harvested, pelleted by centrifugation, and solubilized at 4 8Cin mammalian cell lysis buffer [0.5% Nonidet P-40/20 mM
Tris, pH 7.5/20 mM h-glycerol phosphate/10 mM NaF/0.5
mM Na3VO4/150 mM NaCl/1 mM DTT/1 mM PMSF/0.2
mM EGTA (pH 7.0)/5 Ag/ml pepstatin, leupetin and
chymostatin]. Fifteen minutes later, lysates were clarified
by centrifugation at 4 8C, 10,000 rpm for 30 min. The
supernatant was used in immunoblotting, immunoprecipita-
tion and pulldown experiments. For electrophoretic mobility
shift assay (EMSA) experimets, cells were resuspended in
CE buffer [0.1% Triton X-100/10 mM HEPES (pH 7.9)/5
mM MgCl2/10 mM KCl/1 mM DTT/1 mM PMSF],
incubated on ice for 15 min and pelleted by centrifugation
at 4 8C, 3500 rpm for 10 min. The supernatant represented
the cytoplasmic extract (CE). The pellet was further
resuspended in NE buffer [20 mM HEPES (pH 7.9)/25%
glycerol/0.42 M NaCl/1.5 mMMgCl2/0.2 mM EDTA/1 mM
DTT/1 mM PMSF], incubated on ice for 20 min and
centrifuged at 4 8C, 14,000 rpm for 10 min. The supernatant
represented the nuclear extract (NE). Protein expression was
checked by Western blot. Both cytoplasmic and nuclear
extracts were stored at �70 8C.
2.5. Electrophoretic mobility shift assay (EMSA)
NE extracts were assayed for NF-nB binding to its
cognate nB site exactly according to manufacturer’s
instruction (Promega cat. no. E3050). Briefly, NE extracts
containing equal amounts of whole proteins were incu-
bated with 32P-labeled nB oligonucleotide (AGTT-
GAGGGGACTTTCCCAGGC) in buffer containing 4%
glycerol, 1 mM MgCI2, 50 mM NaCl, 0.5 mM EDTA, 0.5
mM DTT, 10 mM Tris–HCl (pH 7.5), and 0.05 mg/ml
poly (dI–dC)d (dI–dC). Sp1 oligonucleotide was used as
control. After incubating at 4 8C for 30 min, aliquots were
fractionated at 4 8C on 0.5� TBE, 4% native polyacry-
lamide gels. Gels were dried and exposed at �70 8C to
XAR film with intensifying screens. For the competitive
assay, 100-fold excessive amount of cold nB oligodeox-
ynucleotide was added into the binding reaction. For super
shift assay, 0.2 Ag of indicated antibodies was incubated
with other components of the binding reaction as was done
in EMSA.
2.6. Coimmunoprecipitation and GST pulldown
Endogenous InBa and IKK were immunoprecipitated
with their corresponding antibody coupled onto protein A
beads. Flag-tagged proteins were immunoprecipitated with
anti-Flag beads (Sigma F-2426) directly. Soluble cell lysates
containing equal amounts of whole proteins were incubated
on a rotor with the indicated beads at 4 8C for 2–4 h. The
beads were washed extensively three to four times with cell
lysis buffer and once with TBS. The beads were added with
SDS-PAGE loading buffer and resolved by Western blot
using indicated antibodies. Or the beads were used in the
subsequent reactions. For GST pulldown, reaction products
were incubated with Glutathione Sepharose 4B (Pharmacia
Biotech code no. 17-0756-01) in TBS and 1% NP-40 for 2 h
and the Sepharose beads were washed thoroughly with TBS
and 1% NP-40 three to four times. The protein was eluted
by adding sample buffer, subsequently run on 9% SDS-
PAGE gel and visualized by immunoblotting with anti-Ub
antibody.
2.7. In vitro InBa polyubiquitination assay
In vitro translated 35S-InBa or GST-InBa or control
proteins were incubated with purified ubiquitin (0.1 mM),
E1 (0.1 mM), Ubch5A (0.2 mM), and E3 (ICP0, BICP0 or
control proteins) in the reaction buffer containing 50 mM
Tris, pH 7.5, 5 mM MgCl2, 2 mM ATP, 0.1 AM Ubal. The
mixtures were thoroughly mixed and allowed to react at 37
8C for 90 min. For 35S-InBa as substrate, the reaction
products were loaded onto 9% SDS-PAGE gel and auto-
radiograph was obtained. For GST-InBa as substrate, the
L. Diao et al. / Cellular Signalling 17 (2005) 217–229220
reaction products were processed with GST pulldown and
subjected to immunoblotting with anti-Ub antibody.
2.8. In vitro IKK kinase assay
To monitor endogenous IKK activity, IKK complex was
immunoprecipitated by anti-IKK protein A beads from cell
extracts transfected for 24 h with LacZ, ICP0, BICP0 or
TRAF6. The beads were then incubated with GST-InBa and
0.5 ACi g-32P-ATP together with a kinase buffer containing
50 mM Tris–HCl, pH 7.5, 5 mM MgCl2, 50 AM ATP. After
incubating at 30 8C for 1 h, the reaction products were
resolved by SDS-PAGE and autoradiograph was obtained.
3. Results
3.1. ICP0 and BICP0 stimulate NF-nB-dependent tran-
scription in vivo
Initially, we set out to characterize BIV-1 LTR enhancer
that is inducible by BICP0. It came into our attention that
this induction was significantly impaired when a short
stretch of the LTR was deleted, in which a potential nBsequence was identified (Fig. 1). This led us to speculate
that HSV-1 ICP0 and BHV BICP0 stimulated gene
transcription via promoting NF-nB activity. To explore
this hypothesis, we transfected a construct encoding ICP0
into human neuroblastoma SH-SY5Y cell together with a
nB-Luc reporter gene that expresses luciferase under the
control of three tandem repeats of NF-nB binding sites
(Fig. 2a). Furthermore, a plasmid was included as a
negative control to express LacZ in parallel experiments. A
strong stimulation of the nB-Luc was observed when ICP0
was expressed. The folds of stimulation were in direct
proportion to the amount of ICP0 expressed (Fig. 2b). In
contrast, no stimulation was detected when LacZ was
expressed (data not shown). Likewise, we transfected
another construct encoding BICP0 and found that BICP0
Fig. 1. Activation of BIV-LTR by BICP0 is dependent on a potential nB site on the
the right side with the deleted regions marked. BICP0 was co-transfected into 29
these constructs by BICP0 is represented at the left side as fold increase of luciferas
nB site. TAR: Tat-associated region.
stimulated NF-nB-dependent transcription in SY5Y cell in
a similar, but more potent, way (Fig. 2b). Since HSV-1
infects other cell lineages besides neuron, we undertook
the same luciferase assay experiments to check whether
ICP0 and BICP0 could still do so in 293T and HeLa cells.
As was shown in Fig. 2c and d, NF-nB-dependenttranscriptions were indeed promoted by ICP0 and BICP0,
suggesting that this activation induced by ICP0 and BICP0
was independent of cell lines although HSV-1 infection
exhibited cell line dependence.
Previously, ICP0 was proposed to activate gene tran-
scriptions by interfering with ND10 substructure. To rule
out the possibility that NF-nB activation by ICP0 was due to
indiscriminate potentiation of transcription factors in the
nucleus, we transfected LEF-Luc, Gal4-Luc, or nB-Lucreporter genes individually into 293T cell together with
ICP0. As was expected, nB-Luc transcription was markedly
increased by ICP0. However, LEF-Luc and Gal4-Luc gene
transcriptions remained constant in the presence or absence
of ICP0. In contrast, LEF-Luc was activated by its cognate
activator (DN)-h-catenin. The same was true for Gal4-Luc
in response to its cognate activator Gal4-VP16. These
results indicated that ICP0 specifically activated NF-nB-dependent gene transcription (Fig. 3a).
It was reported that the N-terminal part of ICP0
contained a RING domain characteristic of ubiquitin E3.
Likewise, the RING domain is also conserved in BICP0, of
which the cysteine residues at position 13 and 51 were
found to be critical in substrate-independent poly-ubiquiti-
nation reaction (Fig. 3d). To test whether the RING domain
is essential for activating NF-nB, BICP0 or BICP0(13G/
51A) constructs were transfected into 293T cell along with
nB-Luc reporter gene. As was shown in Fig. 3c,
BICP0(13G/51A) achieved little stimulation above back-
ground while BICP0 could induce robust activation of NF-
nB, implicating that the RING domain was absolutely
necessary for NF-nB activation and ubiquitin was possibly
involved in the process. We also tested truncations of ICP0
and BICP0 to explore whether ubiquitin ligase activity alone
enhancer. Diagrams of the BIV-LTR and its truncated mutants are shown at
3T cell with BIV-LTR or its truncated mutants, respectively. Stimulation of
e readout. The solid black bar is used to highlight the existence of a potential
Fig. 2. Both ICP0 and BICP0 can activate NF-nB responsive reporter gene in different cells. (a) Schematic representation of nB-Luc luciferase reporter geneused in this study. Note that the nB sequence of this construct is an authentic one that is slightly different from the potential nB site identified in Fig. 1. ICP0 or
BICP0 was co-transfected into cell along with this reporter gene for 48 h. Shown in (b) was fold inductions of the nB-Luc expression by gradient amount of
ICP0 or BICP0 in Neuroblastoma SH-SY5Y cells. The same luciferase assay experiments were carried out in (c) HeLa cells and (d) 293T cells. Bottom panels:
the gradient expression of ICP0 or BICP0 in 293T cells.
L. Diao et al. / Cellular Signalling 17 (2005) 217–229 221
was sufficient to activate NF-nB. It turned out that either
ICP0(N) or ICP0(C) alone could not activate NF-nB-dependent transcription (Fig. 3b), nor did the BICP0(C).
Although BICP0(N) displayed some stimulation on NF-nB,this was due to the fact that BICP0(N) covered not only the
RING domain, but also a much longer stretch of peptide
located C-terminal to the RING domain as compared to the
ICP0(N), which might mediate protein–protein interactions.
Taken together, these results established that both ICP0 and
BICP0 could specifically activate NF-nB-dependent tran-scription in different cell lines, which required the RING
domain.
Fig. 3. The RING domains of both ICP0 and BICP0 are important for their selective activation of NF-nB. (a) ICP0 activates nB-Luc, but not LEF-Luc or Gal4-Luc reporter genes. ICP0 or control plasmids were co-transfected into 293T cell along with the indicated reporter genes. (DN)-h-catenin and Gal4-VP16 are thecognate activators of LEF-Luc and Gal4-Luc, respectively. (b) nB-Luc activation induced by truncations of ICP0. ICP0(exon1+2): N-terminus of ICP0
harboring the RING domain; ICP0(MC) and ICP0(AC): C-terminal parts of ICP0 that do not contain the RING domain. (c) nB-Luc activation induced by
mutants of BICP0. BICP0(N): N-terminal half of BICP0; BICP0(C): C-terminal half of BICP0; BICP0(13G/51A): C13G and C51A point mutation in the
RING domain. (d) BICP0 can catalyze poly-ubiquitin chain formation. The reaction mixture contained Ubiquitin, E1, Ubch5A and ATP besides BICP0 or
BICP0(13G/51A) as indicated. Poly-Ub chains were detected by anti-Ub antibody.
L. Diao et al. / Cellular Signalling 17 (2005) 217–229222
3.2. ICP0 and BICP0 induce nuclear translocation of p65-
NF-nB and enhance its binding affinity to nB site
Normally, NF-nB is sequestered within cytoplasm and
translocates into nucleus upon activation [26]. To deter-
mine if ICP0 also exploited this mechanism to activate
NF-nB, we investigated whether it could influence the
subcellular distribution of NF-nB. So ICP0, BICP0,
BICP0(13G/51A) and LacZ were transfected into 293T
cell, respectively. After 16 h, the cells were immunos-
tained in accordance with the manufacturers’ recommen-
dations and imaged by Bio-Rad confocal fluorescent
microscope. The distribution of endogenous p65-NF-nBwas revealed by anti-p65 antibody in combination with
FITC-conjugated secondary antibody. Obviously, p65 was
distributed diffusely at the circumference of the cell when
LacZ was transfected, which reflected the stationary state
of NF-nB. In contrast, ICP0 and BICP0 induced p65 to
concentrate in the center of the cell, which was superposed
neatly with the nucleus as revealed by DAPI staining to
the nucleus. This indicated that both ICP0 and BICP0
could drive translocation of p65-NF-nB from cytoplasm
into nucleus. In addition, BICP0(13G/51A) failed to cause
any change to the subcellular distribution of p65, which
again suggested that the RING domain was indispensable
(Fig. 4a).
Another consequence of NF-nB activation is the specific
targeting of NF-nB to its cognate nB sites. To verify that
ICP0 induced the same effect, nuclear extracts were
prepared from 293T cell transfected with either ICP0 or
LacZ for different lengths of time and then incubated with
radioactive synthetic nB oligodeoxynucleotide. Gel shift
assay indicated that a new activity was elicited in the
nucleus in response to ICP0 that could bind tightly to the nB
Fig. 4. ICP0 and BICP0 induce nuclear translocation of NF-nB-p65 and enhance its binding affinity to nB site. (a) ICP0 and BICP0 induce p65-NF-nB to
translocate from cytoplasm into nucleus. ICP0 or control plasmids were transfected into 293T cell. After 16 h, the cells were immunostained and imaged as
described in Section 2. Green: p65; dark blue: nucleus; white: p65 in nucleus; pink: p65 not inside nucleus. (b) ICP0 induces an activity that can bind to
radioactive nB oligonucleotides as revealed by electrophoresis mobility shift assay (EMSA). (c) The EMSA band can be competed away by 100-fold excess of
cold nB oligonucleotides. (d) Addition of anti-p65 antibody to the EMSA reaction mixture can cause a supershift band relative to the normal shift band.
L. Diao et al. / Cellular Signalling 17 (2005) 217–229 223
L. Diao et al. / Cellular Signalling 17 (2005) 217–229224
probe, while LacZ was unable to do so (Fig. 4b).
Furthermore, this activity did not bind to radioactive
synthetic sp1 oligodeoxynucleotide (Data not shown). More
experiments showed that BICP0 could also induce this
activity, while BICP0(13G/51A) was deprived of this
function (Fig. 4c). In order to confirm that the new activity
targeted only the nB oligodeoxynucleotide, 100-fold exces-
sive amount of cold nB probe was added into the binding
reaction. As a result, the cold nB probe competed away all
the ICP0 or BICP0 responsive proteins and led to the
complete diminishing of gel shift bands representing protein
and DNA interaction, demonstrating that the activity
induced by ICP0 or BICP0 recognized nB sequence
specifically (Fig. 4c). Finally, the identity of the induced
activity was confirmed by supershift EMSA. When antibody
specific to h-actin was incubated with nB probe and nuclear
extracts transfected with ICP0 or BICP0, the resulting gel
shift bands ran at the same position as those of the binding
reaction products without adding any antibody. In contrast,
when antibody specific to p65-NF-nB was introduced, the
resulting gel shift bands apparently ran more slowly than
those of the control (Fig. 4d). These proved that both ICP0
and BICP0 were able to cause NF-nB binding specifically to
its cognate nB promoter.
3.3. ICP0 interacts with InBa and its stimulatory effect is
attenuated by Ubch5A(C85A) and MG132
So far, it was well established that both ICP0 and BICP0
had the ability to stimulate NF-nB. We therefore went on to
investigate how they activated this signaling pathway. At
first, we found that ICP0 could not stimulate IKK activity as
revealed by IKK kinase assay (Fig. 5a). It was known that
when Serine 32 and 36 of InBa were all mutated into
alanine, this mutant InBa(S32/36A) became a super-
repressor that could block NF-nB activation by many NF-
nB specific stimuli. For example, TRAF6, an upstream
activator of IKK, could activate NF-nB and this activation
was severely inhibited by InBa(S32/36A). Consequently,we explored whether InBa(S32/36A) had the same effect on
NF-nB activation mediated by ICP0. When ICP0 and nB-Luc were transfected into 293T cell with or without
InBa(S32/36A), NF-nB activation by ICP0 was not affected
at all even in the presence of a large amount of InBa(S32/36A), which again suggested that InBa phosphorylation
was not essential for ICP0-mediated NF-nB activation and
ICP0 acted downstream of IKK, probably targeting InBadirectly (Fig. 5b).
Like InBa (S32/36A), MG132 was a potent inhibitor of
NF-nB signaling pathway by specifically interfering with
the action of 26S proteasome. To determine whether this
was true for ICP0, 293T cell was transfected with ICP0 and
nB-Luc for 24 h, after which MG132 or control reagents
were added into the culture medium and cells were
harvested after an additional 8 h. Luciferase assay revealed
that while DMSO, aprotinin, chymostatin and leupeptin had
no inhibitory effects on ICP0-mediated NF-nB activation,
MG132 produced significant reduction of NF-nB activation
in a dose-dependent manner. This indicated that protein
degradation was required during NF-nB activation and ICP0
followed the canonical NF-nB pathway at the terminal part
(Fig. 5c).
Ubch5A is a kind of ubiquitin conjugating enzyme E2,
and a point mutation at residue 85 from cysteine to alanine
abolished its proper activity completely. In combination
with it, ICP0 was previously shown to catalyze poly-
ubiquitination of p53 [17]. In addition, Ubch5A was
implicated to be an important E2 during InBa ubiquitination
by its endogenous E3, the SCF complex [29]. Therefore, we
investigated whether Ubch5A was important for NF-nBactivation by ICP0. ICP0 and nB-Luc were transfected into
293T cell with or without the mutant Ubch5A(C85A).
Luciferase assay revealed that Ubch5A(C85A) significantly
reduced NF-nB activation elicited by ICP0. In contrast,
another E2 mutant Ubch13(C87A) had no inhibitory effect
on NF-nB activation by ICP0, although this mutant had lost
its catalytic activity too (Fig. 5d).
Based on the above functional analysis, it is very likely
that ICP0 may be integrated into the NF-nB signaling
pathway at the InBa site. To explore this possibility, we co-
transfected flag-ICP0 with IKKh or a series of HA-InBamutants, respectively. Then flag-ICP0 immunoprecipitates
were analyzed to check which protein was pulled down by
ICP0. It turned out that full-length InBa interacted
specifically with ICP0, while IKKh did not show any
affinity to ICP0. In addition, ICP0 interacted with InBa(54–317) and it did not interacted with either InBa(1–225) orInBa(1–275), which suggested that ICP0 recognized the C-
terminus of InBa (Fig. 5e). Taken together, these results
suggested that ICP0 might shortcut the NF-nB signaling
pathway and target InBa directly. Importantly, it implicated
that ICP0 might serve as an ubiquitin ligase E3 in the
process.
3.4. ICP0 and BICP0 catalyze InBa ubiquitination in vivo
and in vitro
In light of the facts that ICP0 was an ubiquitin ligase E3
and it interacted with InBa in vivo, we wondered whether
ICP0 could catalyze the poly-ubiquitination reaction with
InBa as the substrate. To address this hypothesis, we set up
an in vitro reaction system that contained in vitro translated35S-InBa and the purified E1 and E2 components of the
ubiquitin system. When incubating them together, no
modification of InBa was observed. Interestingly, when
ICP0 or BICP0 was introduced into the reaction mixture, a
ladder of InBa species was generated with the molecular
weight spanning from about 40 kDa up to more than 200
kDa, which suggested that InBa was poly-ubiquitinated in
the presence of ICP0 or BICP0. In contrast, neither the
mock bead nor various ICP0 truncation mutants could help
produce such high molecular weight species of InBa. In
Fig. 5. ICP0 interacts with InBa and its function is attenuated by Ubch5A(C85A) and MG132. (a) ICP0 does not stimulate IKK activity. After transfecting
293T cell with the indicated constructs for 24 h, endogenous IKK was immunoprecipitated. Upper panel: IKK activity was checked by standard kinase assay
using bacteria expressed InBa as substrate. Lower panel: equal efficiency of IKK immunoprecipitation. (b) Activation of NF-nB by ICP0 is not inhibited by
InBa(S32/36A). Upper panel: nB-Luc activation by ICP0 or TRAF6 in the presence of gradient amounts of InBa(S32/36A); lower panels: expression of the
indicated constructs. (c) Activation of NF-nB by ICP0 is inhibited by MG132. ICP0 was transfected into 293T cells together with nB-Luc reporter gene. After24 h, cells were mock-treated or treated with MG132, aprotinin, chymostatin or leupeptin at indicated final concentrations. Luciferase activity was measured 8
h later. (d) NF-nB activation by ICP0 is attenuated by Ubch5A(C85A). (e) ICP0 physically interacts with InBa. Flag-ICP0 was co-transfected into 293T cells
with the indicated constructs. ICP0 was immunoprecipitated by anti-flag beads. The beads were checked with anti-HA or anti-IKK antibody.
L. Diao et al. / Cellular Signalling 17 (2005) 217–229 225
addition, BICP0(13G/51A) was unable to catalyze the
modification of InBa, which was consistent with the fact
that BICP0(13G/51A) harbored two point mutations at its
RING domain and it did not possess any ubiquitin E3
activity. As a further control, TRAF6 was also a demon-
strated ubiquitin E3 working at the far upstream of the NF-
nB signaling pathway. However, it failed to catalyze the
modification of InBa when it was added into the reaction
mixture (Fig. 6a). These results indicated that both ICP0 and
BICP0 were responsible for the specific modification of
InBa.
In the previous section, we found that activation of NF-
nB by ICP0 was independent of the phosphorylation of
InBa at either Ser32 or Ser36. Therefore, we continued to
investigate whether this phosphorylation was important for
InBa modification in this context. Consistently, ICP0
promoted indiscriminately the modification of InBa(WT),
InBa(S32A), InBa(S36A) and InBa(S32/36A) to the same
extent (Fig. 6b). These results again supported the notion
that ICP0 targeted InBa directly.
Next was studied the identity of the InBa modification
caused by ICP0 and BICP0. For the sake of protein
Fig. 6. ICP0 and BICP0 catalyze InBa ubiquitination in vivo and in vitro, independent of InBa phosphorylation. (a) ICP0 and BICP0 catalyze modification of35S-InBa. ICP0 or control proteins were incubated with 35S-InBa and purified components of ubiquitin system. The product was resolved with SDS-PAGE and
autoradiographed. (b) The modification of InBa induced by ICP0 is independent of Serine 32/36 phosphorylation. WT: wild type; S32A or S36A: serine 32 or
36 was substituted by alanine; S32/36A: both serines 32 and 36 were changed to alanines. (c) ICP0 and BICP0 catalyze poly-ubiquitination of GST-InBa. Invitro ubiquitination reactions were performed with GST-InBa as substrate and GST or GST-ATF as a control. The reaction products were pulled down with
Glutathione Sepharose beads and probed with anti-Ub antibody. (d) ICP0 and BICP0 catalyze poly-ubiquitination of InBa in an E1-, E2- and Ub-dependent
way. Experiment was carried out as in (c) except that a specific component was left out for each lane. (e) ICP0 and BICP0 stimulate poly-ubiquitination of
InBa in vivo. 293T cell was transfected with indicated constructs for 24 h. Endogenous InBa was immunoprecipitated after treating cell with MG132 for an
additional 8 h.
L. Diao et al. / Cellular Signalling 17 (2005) 217–229226
precipitation and Western blot, we used as substrate GST-
InBa purified from bacteria instead of in vitro translated35S-InBa in the following experiments. Once the modifica-
tion reactions were over, the GST-InBa and the control
substrates were pulled down and washed extensively. Then
the products were loaded onto SDS denaturing gel and
probed with antibody specifically against ubiquitin. As was
expected, GST-InBa from reactions containing ICP0 or
BICP0 produced characteristic smears of ubiquitin species
representing different numbers of ubiquitins conjugated
with the GST-InBa. However, neither TRAF6 nor
BICP0(13G/51A) was able to cause GST-InBa to generate
such fingerprint on gel that was detectable by ubiquitin
antibody. Furthermore, GST-ATF and GST alone from
reactions containing ICP0 did not show any characteristic
smears that were recognized by ubiquitin antibody (Fig. 6c).
These data conclusively established that the modified InBaby both ICP0 and BICP0 were indeed poly-ubiquitinated
species of InBa.This conclusion could also be substantiated by examin-
ing contribution of the components in ubiquitin system to
the poly-ubiquitination of InBa. If purified ubiquitin,
ubiquitin activating enzyme E1, ubiquitin conjugating
enzyme Ubch5A or ICP0 was left out one item at a time,
the characteristic smears of the modified InBa disappeared
completely when probing it with ubiquitin antibody.
Consistently, the inactive Ubch5A(C85A) could not help
the generation of the InBa poly-ubiquitin either. Only when
all the components were incubated together could the poly-
ubiquitinated forms of InBa be produced (Fig. 6d). These
L. Diao et al. / Cellular Signalling 17 (2005) 217–229 227
data clearly showed that ICP0 and BICP0 were ubiquitin
ligase E3 for InBa.Finally, we investigated whether endogenous InBa was
modified by ubiquitin in response to ICP0 and BICP0.
Therefore, 293T cells were transfected with ICP0, BICP0,
BICP0(13G/51A), LacZ or pcDNA3.1, respectively.
Twenty-four hours later, MG132 was added into culture
medium to prevent the de-ubiquitination of InBa and the
cells were harvested after an additional 8 h. Endogenous
InBa was immunoprecipitated with InBa specific antibody
coupled onto protein A bead and the immunoprecipitates
were probed with antibody specifically against ubiquitin. It
turned out that immunoprecipitates from BICP0(13G/51A),
LacZ or pcDNA3.1 transfected cells did not contain proteins
that could be detected by ubiquitin antibody, while the
endogenous InBa was among the immunoprecipitates.
Strikingly, the immunoprecipitates from either ICP0 or
BICP0 transfected cells did contain InBa smears that were
clearly recognized by ubiquitin antibody (Fig. 6e). These
results indicated that both ICP0 and BICP0 could catalyze
InBa poly-ubiquitination in vivo.
4. Discussion
There are several means by which HSV-1 can affect the
life cycle of human immunodeficiency virus type I (HIV-1)
[32]. One possible way involved stimulation of HIV-1 LTR
enhancer by the immediate-early protein ICP0 encoded in
HSV-1, which consequently enhanced gene expressions and
replication of HIV-1 per se [33]. However, the mechanism
underlying this trans-activation is poorly understood. One
recent study [34] suggested that ICP0 could functionally
cooperate with HIV Tat to activate LTR even in the absence
of HIV TAR sequence. They proposed that ICP0 recruited
Tat to the vicinity of the LTR enhancer to achieve this end.
Contradictorily, they did not show any physical interaction
between ICP0 and Tat either in vivo or in vitro. Signifi-
cantly, ICP0 has never been shown to bind to DNA in vivo
or in vitro. It seems unlikely that ICP0 functions as a
transcription co-factor in this process. In fact, current model
of ICP0 action favors an indirect mechanism in promoting
transcription of both viral and cellular genes [5]. Previously,
it has been demonstrated that activation and binding of NF-
nB at the HIV LTR is required for and can support enhanced
LTR-mediated transcription in response to cytokines such as
TNF-a and IL-1h, lectins, PMA and bacterial LPS, etc.
[35]. In this study, we found that BICP0, the ICP0
homologue from bovine herpes virus, could robustly induce
activation of the LTR from bovine immunodeficiency virus.
This induction was not affected when the TAR sequence of
the BIV was removed. In contrast, this activation was
severely impaired when a short stretch of DNA upstream to
the U3 segment was deleted, in which a nB consensus
segment was identified. This observation implicated an
alternative model of ICP0 and BICP0 action and led us to
hypothesize that both ICP0 and BICP0 could activate NF-
nB signaling pathway.
This hypothesis is supported by several lines of
evidence in the present study. First, both ICP0 and BICP0
were able to significantly stimulate gene expression of a
luciferase reporter construct that contained nothing derived
from the LTR and was introduced instead with three
tandem repeats of authentic nB sequence at the promoter
region. In addition, ICP0 could only activate this nB-Lucreporter construct and had no stimulatory effect on either
LEF-Luc or Gal4-Luc reporter construct. Notably, the LEF
binding site was previously identified inside the LTR and
it was functionally implicated in the potentiation of the
LTR by the Wnt signaling pathway [36]. Therefore, a
functional connection could be drawn between ICP0 and
the NF-nB signaling pathway. Second, both ICP0 and
BICP0 could induce translocation of p65-NF-nB from
cytoplasm into nucleus. In addition, NF-nB from ICP0- or
BICP0-treated nuclear extract was able to bind specifically
to its cognate nB site as revealed by EMSA assay, which
clearly indicated that NF-nB was activated, due to the
release of NF-nB from its inhibitor InBa, in response to
ICP0 and BICP0. This possibility was further substantiated
by the finding that activation of NF-nB by ICP0 was
specifically inhibited by proteasome inhibitor MG132,
suggesting that degradation of InBa was essential for the
process and NF-nB activation followed the classical
mechanism in this context. Third, ICP0 was found to
interact with the C-terminus of InBa and it did not
interact with IKKh, the kinase specific for phosphorylat-
ing InBa, which suggested that ICP0 might shortcut the
NF-nB signaling pathway and integrate itself into this
pathway at the InBa point. This conjecture was supported
by the experimental result that the super-repressor
InBa(S32/36A) exhibited no inhibitory effect on NF-nBactivation by ICP0, although this super-repressor was
proved to be a strong inhibitor of NF-nB activation by
many stimuli (e.g., TRAF6) in which phosphorylation of
InBa was an essential step. Consistently, ICP0 did not
influence the kinase activity of IKK complex when ICP0
was expressed inside the cell. Fourth, the RING domains
of both ICP0 and BICP0 were necessary for NF-nBactivation and the base-point mutant of BICP0(13G/51A)
was deprived of the ability to stimulate NF-nB with
respect to the criteria explored in this study. In addition,
the ubiquitin E2 mutant Ubch5A(C85A) was found to
attenuate NF-nB activation in response to ICP0. Given
that ICP0 is an ubiquitin ligase E3 and InBa is poly-
ubiquitinated and degraded before NF-nB activation, we
demonstrated in this investigation that ICP0 and BICP0
could catalyze conjugation of ubiquitin onto InBa,independent of its phosphorylation status. Furthermore,
this process was absolutely dependent on each component
of the ubiquitin system and could be confirmed in vivo by
monitoring endogenous InBa poly-ubiquitination in
response to ICP0 and BICP0.
L. Diao et al. / Cellular Signalling 17 (2005) 217–229228
Although activation of NF-nB in response to microbial
stimuli is normally associated with the initiation of humoral
and cellular immunity, some pathogens have taken advant-
age of this system to enhance their own replication, survival
and dissemination within the host [37]. For example, HTLV-
1 virus is a causative microbial capable of transforming T
cell and is responsible for adult T-cell leukemia. It has been
shown that the oncoprotein Tax from HTLV-1 could interact
with both IKK and another NF-nB inhibitor p100, thus
promoting phosphorylation and degradation of this inhib-
itor. However, Tax itself did not possess any enzymatic
activity. It activated NF-nB by bringing the IKK close to its
substrate and leaving the cellular components to finish the
job [38]. In contrast, ICP0 and BICP0 are ubiquitin ligase
E3 and it could directly catalyze InBa poly-ubiquitination.
Therefore, phosphorylation of InBa became a redundant
process for ICP0 and BICP0 function and Herpes virus
chose a more economical way to activate NF-nB. As an
ubiquitin ligase, ICP0 was previously connected to the
degradation of promyelocytic leukemia antigen (PML) and
Sp100 in ND10 [9,39]. Recently, ICP0 was also shown to
interact with tumor suppressor p53 and catalyze ubiquitina-
tion of the latter [17]. Although the multifunctionality of
ICP0 could be understood in reference to its ubiquitin ligase
activity, it is still a great challenge to integrate the seemingly
disparate targets of ICP0 action in terms of its overall
contribution in HSV-1 life cycle.
In summary, our study identified a new signaling
pathway that ICP0 hijacked to activate a myriad of viral
and cellular genes: when ICP0 is expressed immediately
after HSV-1 initial infection or reactivation, it specifically
recognizes and interacts with the C-terminus of InBa. Incombination with ubiquitin E1 and Ubch5A, ICP0 catalyzes
conjugation of one ubiquitin after another onto InBadirectly. In turn, the poly-ubiquitinated InBa is earmarked
and degraded by the 26S proteasome. This releases NF-nBfrom its cytoplasm anchor and it is free to enter the nucleus
and turns on its target genes, including those regulated by
the LTR enhancer. Interestingly, a nB consensus site was
previously identified in the promoter region of ICP0 gene in
HSV-1. In addition, NF-nB was shown to recognize and
bind this sequence [40]. In light of the new mechanism of
ICP0 action presented here, it is possible that there exists a
positive feedback regulation in ICP0 gene expression in
which more ICP0 can be produced from a tiny amount of
ICP0 via NF-nB signaling pathway. It is known that cellular
stress can induce HSV-1 to exit from the latent phase of its
life cycle and the same signal can activate NF-nB signaling
pathway too. During HSV-1 phase transition, ICP0 plays an
important role in regulating the virus gene expression. But
how, in the first place, is the ICP0 induced in response to
stress? It is likely that stress alone primes NF-nB signaling
pathway, which in turn induces ICP0 expression from HSV-
1. Then the induced ICP0 can augment this effect by
producing much more ICP0 via shortcutting NF-nBactivation directly. As a result, HSV-1 gene program is
turned on and more virus progenies are produced. This
regulation may be important during HSV-1 reactivation
from latency. More experiments are needed to explore this
wonderful model of HSV-1 reactivation.
Acknowledgement
We thank Professors Tom Gilmore(Boston University,
USA), Allan Weissman (National Institute of Health, USA),
Mike Ellison(University of Alberta, Canada), James Chen
(University of Texas Southwestern Medical Center, USA)
for providing plasmids in this study. We are grateful to
professor Lin Li and Youxin Jin for technical help. Naihe Jin
was supported by the National Key Basic Research and
Development Program (2002CB713802). Yunqi Geng was
supported by National Natural Science Foundation
(30170038). Chen Wang was a scholar of bThe Rising Star
ProgramQ from Shanghai Municipal Government and of
bThe Hundred Talents ProgramQ from Chinese Academy of
Sciences. This work was supported in part by bTheDistinguished Young Scholars ProgramQ from National
Natural Science Foundation of China (30225013), CAS
renovation program and 973 Project (2002CB513003).
References
[1] E. Wagner, D. Bloom, Clin. Microbiol. Rev. 10 (1997) 419–443.
[2] R. Whitley, B. Roizman, Lancet 357 (2001) 1513–1518.
[3] E. Wagner, J. Guzowski, J. Singh, Prog. Nucleic Acid Res. Mol. Biol.
51 (1995) 123–165.
[4] C. Jones, Clin. Microbiol. Rev. 16 (2003) 79–95.
[5] R. Everett, BioEssays 22 (2000) 761–770.
[6] W.P. Halford, P.A. Schaffer, J. Virol. 75 (2001) 3240–3249.
[7] R. Jordan, P. Schaffer, J. Virol. 71 (1997) 6850–6862.
[8] R. Everett, G. Maul, EMBO J. 13 (1994) 5062–5069.
[9] R.D. Everett, P. Freemont, H. Saitoh, M. Dasso, A. Orr, M. Kathoria,
J. Parkinson, J. Virol. 72 (1998) 6581–6591.
[10] G. Maul, Bioessays 20 (1998) 660–667.
[11] R.D. Everett, M. Meredith, A. Orr, A. Cross, M. Kathoria, J.
Parkinson, EMBO J. 16 (1997) 1519–1530.
[12] Y. Kawaguchi, C. Van Sant, B. Roizman, J. Virol. 71 (1997)
7328–7336.
[13] Y. Kawaguchi, R. Bruni, B. Roizman, J. Virol. 71 (1997) 1019–1024.
[14] Y. Kawaguchi, M. Tanaka, A. Yokoymama, G. Matsuda, K. Kato, H.
Kagawa, K. Hirai, B. Roizman, PNAS 98 (2001) 1877–1882.
[15] F. Yao, P. Schaffer, J. Virol. 68 (1994) 8158–8168.
[16] C. Boutell, S. Sadis, R.D. Everett, J. Virol. 76 (2002) 841–850.
[17] C. Boutell, R.D. Everett, J. Biol. Chem. 278 (2003) 36596–36602.
[18] R. Hagglund, B. Roizman, PNAS 99 (2002) 7889–7894.
[19] R. Hagglund, C. Van Sant, P. Lopez, B. Roizman, PNAS 99 (2002)
631–636.
[20] A. Weissman, Nat. Rev., Mol. Cell Biol. 2 (2001) 169–178.
[21] C. Pickart, Annu. Rev. Biochem. 70 (2001) 503–533.
[22] C. Joazeiro, A. Weissman, Cell 102 (2000) 549–552.
[23] L. Hicke, Nat. Rev., Mol. Cell Biol. 2 (2001) 195–201.
[24] C. Wang, L. Deng, M. Hong, G. Akkaraju, J. Inoue, Z. Chen, Nature
412 (2001) 346–351.
[25] V. Dixit, T. Mak, Cell 111 (2002) 615–619.
[26] S. Ghosh,M.May, E. Kopp, Annu. Rev. Immunol. 16 (1998) 225–260.
L. Diao et al. / Cellular Signalling 17 (2005) 217–229 229
[27] M. Karin, Y. Ben-Neriah, Annu. Rev. Immunol. 18 (2002) 621–663.
[28] H. Pahl, Oncogene 18 (1999) 6853–6866.
[29] E. Spencer, J. Jiang, Z.J. Chen, Genes Dev. 13 (1999) 284–294.
[30] D. Finley, Nature 412 (2001) 283–286.
[31] J. Hiscott, H. Kwon, P. Genin, J. Clin. Invest. 107 (2001) 143–151.
[32] G. Palu, L. Benetti, A. Calistri, Herpes 8 (2001) 50–55.
[33] J.M. Ostrove, J. Leonard, K.E. Weck, A.B. Rabson, H.E. Gendelman,
J. Virol. 61 (1987) 3726–3732.
[34] S. Schafer, J. Vlach, P. Pitha, Virologie 70 (1996) 6937–6946.
[35] M. Bate, S. Jassal, D. Brighty, MethodsMol. Biol. 99 (2000) 277–295.
[36] P. Sheridan, C. Sheline, K. Cannon, M. Voz, M. Pazin, J. Kadonaga,
K. Jones, Genes Dev. 9 (1995) 2090–2104.
[37] C.M. Tato, C.A. Hunter, Infect. Immun. 70 (2002) 3311–3317.
[38] G. Xiao, M.E. Cvijic, A. Fong, E.W. Harhaj, M.T. Uhlik, M.
Waterfield, S.C. Sun, EMBO J. 20 (2001) 6805–6815.
[39] M. Chelbi-Alix, H. de The, Oncogene 18 (1999) 935–941.
[40] B. Rong, T. Libermann, K. Kogawa, S. Ghosh, L. Cao, D. Pavan-
Langston, E. Dunkel, Virology 189 (1992) 750–756.